Many proteins and hormones, synthesized as precursors, are processed into their mature forms by highly-specific proteolytic enzymes of the prohormone convertase family. This family of mammalian endoproteases carries out intracellular cleavage at the COOH-terminal side of dibasic sites within their substrate polypeptides. Members of this prohormone convertase (PC) family are Ca.sup.++ -dependent serine proteases related to the yeast dibasic-specific endoprotease Kex2 (Smeekins, S.P., Bio/Technology 11: 182-186, 1993). Moreover, the catalytic domains thereof are organized similarly to bacterial subtilisins. At least six mammalian prohormone convertases have been found including PC2, PC3/PC1, PC4, PC5/6 furin/PACE and PACE4 (Smeekins, S.P., ibid., Seidah, N.G. et al., Biochimie (France) 76: 197-209, 1994).
Mammalian prohormone convertases act on a wide variety of precursor molecules having an array of biological activities. The proinsulin prohormone was the first substrate precursor identified. Subsequently, over 150 substrates have been found in organisms from yeast to mammals; including neuropeptides, peptide hormones, growth factors and their receptors, plasma and coagulation proteins, retroviral envelope proteins and cellular toxins, e.g., anthrax. Cleavage sites include basic amino acids, with cleavage occurring at paired basic residues, usually after Lys-Arg or Arg-Arg and more rarely Arg-Lys or Lys-Lys (Smeekins, S.P., ibid.).
The prohormone convertase family exhibits tissue-specific expression and cellular compartmentalization, which may relate to biological function. For example, PC1/3 and PC2 are solely expressed in neuroendocrine tissues. Biological activities of these proteins are localized to the regulated secretory pathway in neuroendocrine cells, specifically in secretory granules; and both play important roles in processing precursors in the granules such as proglucagon, proopiomelanocortin (POMC) and proinsulin. Thus, intracellular localization and tissue specificity appear to reflect where the biological activities of these endoproteases are manifested.
PC4 exhibits highly specific tissue-selectivity of gene expression. PC4 has been isolated from mice and rats and is solely expressed in the testis. In mice, PC4 gene expression occurs around the 20th day of gestation corresponding with the first stages of spermatogenesis. High PC4 mRNA expression levels are found in germ cells but not in Leydig, Sertoli, or peritubular cells. In situ hybridization demonstrates mRNA expression in the pachytene spermatocytes and the round spermatids, but not in elongating spermatids (N.G. Seidah et al., Mol. Endocrinol. 6: 1559-1570, 1992). Moreover, in both rat and mouse, three PC4 mRNAs are observed; these RNAs are probably derived from differential splicing and/or exon skipping events. The biological function of murine and rat PC4 proteins derived from the principle or alternatively spliced forms is unknown.
Spermatogenesis is a sequential process taking place in the seminiferous tubules, where germ (or sperm) cells ultimately mature into spermatozoa. Peptides produced within the testis are potential paracrine and autocrine factors mediating interactions between testicular cells. Most of these known potential peptide substrates are produced within Leydig and Sertoli cells which are non-germ cells. Sertoli cells, located within the seminiferous tubules, are in contact with the germ cells and may directly produce testis-specific factors that influence germ cell maturation. Other factors that influence germ cells may be paracrine or endocrine factors; many of these molecules produced outside the seminiferous tubules are transported into the germ cell microenvironment by transport and binding proteins expressed by the Sertoli cells. In addition, paracrine factors that cross the cellular barrier and enter the sperm cell microenvironment include molecules secreted from Leydig cells. Leydig cells are located in the interstitial space found between the seminiferous tubules, and produce several factors that may play important roles in the maturation process, such as testosterone, Leydig factor, IGF-1, inhibin and prohibin. These and other factors may act specifically during a defined stage in the spermatogenic cycle. Moreover, some peptide hormones expressed in germ cells, located in close proximity to Sertoli cells, are potential paracrine and autocrine factors that mediate interactions between testicular cells. For example, the opioid peptides, POMC and proenkephalin, are expressed and presumably processed in germ cells. Interestingly, murine proenkephalin has a similar mRNA expression profile to murine PC4 during spermatogenesis (S. Torii, et al., FEBS Let. 316: 12-16, 1993). The stage-specific expression of murine PC4 implicates a biological role in processing prohormone factors from the testis. Although a role for PC4 in spermatogenesis is suggested, the function of PC4 is unknown.
Thus, the human homologue is sought. The present invention advantageously provides the isolation of a human homologue to murine PC4.